The synapse represents a specialized structure for communication between neurons in the central nervous systems. Members of the ligand-gated ion channel superfamily of neurotransmitter receptors are responsible for rapid transmission of excitatory and inhibitory signals at synaptic sites and their localization at postsynaptic sites is vital for efficient synaptic transmission. The postsynaptic sites are characterized by dense accumulations of submembranous cytoskeletal elements. The mammalian protein gephyrin is crucial for the clustering of inhibitory glycine and GABAA receptors. Gephyrin anchors glycine receptors to the cytoskeleton through simultaneous binding to the 13-subunit of the receptor and tubulin. In addition, gephyrin interacts with other proteins presumably playing important roles in the assembly of postsynaptic densities, including collybistin, RAFT1, profilin and GABARAP. Gephyrin has been postulated to form a hexagonal scaffold underneath the postsynaptic membrane, which provides binding sites for the receptors and elements of the cytoskeleton. The overall goal of this proposal is to evaluate and expand this scaffolding model. One underlying hypothesis is that the functions of gephyrin pertaining to the organization of the postsynaptic membrane are distributed throughout its primary sequence and are not only confined to the linker region as has been generally assumed. This strategy would allow gephyrin to simultaneously engage in multiple binding interactions, thus modulating the activities of several of its binding partners. A second hypothesis of this proposal is that binding of the partner proteins influences the oligomeric state of gephyrin and consequently its ability to form the hexagonal scaffold underneath the postsynaptic membrane. In order to investigate the scaffolding model, gephyrin as well as its complexes will be analyzed by biochemical and crystallographic techniques in order to understand its functional diversity. Specifically, the proposal will identify regions in gephyrin responsible for recognition of its binding partners. The strengths of the protein-protein complexes and their oligomeric states will be analyzed by biophysical techniques. These studies will be complemented by crystal structure analyses of full-length gephyrin, its E-domain and the various protein-protein complexes formed by this protein. These experiments will advance the understanding of the multiple functions of gephyrin in organizing the postsynaptic membrane at inhibitory synapses and will test and extend the scaffolding model of gephyrin.